Neurotransmitter release is acutely triggered by Ca2+ and is regulated during presynaptic plasticity processes that underlie some forms of information processing in the brain. Characterization of the mechanisms of release and its regulation is thus critical to understand brain function and will facilitate the development of therapies for multiple neurological disorders (e.g. schizophrenia, Alzheimer's and Parkinson's) and for diseases involving defects in regulated secretion, which controls many important physiological functions (e.g. heart rate, blood pressure and insulin release). This research is also relevant to cell biology in general because of its importance to understand intracellular membrane fusion. The machinery that controls release contains a core formed by SNARE proteins, Munc18, Munc13, NSF and SNAP, and specialized proteins that regulate release including Munc13 itself, synaptotagmin, complexin, RIM, Rab3 and CAPS among others. The research proposed in this application involves an interdisciplinary approach integrating structural studies of these proteins, reconstitution assays and electrophysiological analyses of neurotransmitter release in neurons performed by collaborators. This research will build on the previous success of this approach, which has yielded many of the three-dimensional structures of the proteins that govern neurotransmitter release, has revealed crucial mechanistic concepts in the field and has allowed reconstitution of basic steps of Ca2+-evoked synaptic vesicle fusion with eight central components of the release machinery. However, despite these advances, the mechanism of release is still unclear and fundamental questions remain unanswered. The ultimate goals of this proposal are to develop a detailed picture of the mechanism of release that integrates the functions of all these proteins and to provide novel insights into how release is regulated in diverse presynaptic plasticity processes. For this purpose, structural studies of complexes formed by these proteins using a combination of biophysical techniques, including cryo-electron microscopy, cryo-electron tomography, NMR spectroscopy and X-ray crystallography, will be performed. A key aspect of these studies, which is essential to make major, definitive advances in this field, will be to analyze protein complexes between two membranes, as the membranes form intrinsic part of the fusion apparatus and thus are expected to have a strong influence on how the protein components are arranged to induce membrane fusion. The structural studies will be complemented with reconstitution experiments to understand how the different proteins control docking, priming and fusion, as well as with physiological analyses performed by collaborators that will test the relevance of the structural and reconstitution studies. This research is expected to establish fundamental principles on neuronal communication that are vital for brain function.